DNA double-strand breaks (DSBs) pose a serious threat to cell viability and genome stability. The two major DSB repair pathways that have been studied extensively are homologous recombination (HR) and non- homologous end-joining (NHEJ). Understanding the detailed regulation of DNA repair pathway choice will help us design better therapy for cancer patients. For example, a major breakthrough in the targeted treatment of BRCA1-mutant cancers is the use of PARP inhibitors (PARPi), which display synthetic lethality with BRCA1 deficiency. Unfortunately, resistance to PARPi therapy is a major clinical problem in BRCA1-mutant cancers. Recent studies showed that loss of another DNA repair protein, 53BP1 renders these BRCA1-deficient cells or tumors HR repair capacity and resistant to PARPi, suggesting that 53BP1 and BRCA1 compete with each other to influence the pathway choice for DSB repair. Since 53BP1 lacks enzymatic activities that would be directly implicated in DNA repair, we thus decided to further explore the 53BP1-dependent pathway, focusing on the identification of downstream effector proteins that directly participate in DNA repair. Excitingly, we uncovered a nuclease Artemis as a PTIP-binding protein. Artemis has endonuclease activity and is a known component of the NHEJ pathway. Our preliminary data demonstrated that Artemis is a major nuclease that acts downstream of 53BP1- dependent DNA repair pathway. Just like 53BP1, loss of Artemis expression in BRCA1-deficient cells leads to PARPi resistance. However, several important questions remain unanswered. We need to further elucidate the underlying mechanisms how the 53BP1-dependent pathway acts in DNA repair and antagonizes HR functions. Importantly, how to effectively treat BRCA1-deficient tumors that become resistant to PARPi is an urgent clinical question. The following Specific Aims are proposed to address these important questions. Aim 1. Determine the mechanism by which 53BP1-dependent NHEJ repair pathway counteracts HR repair pathway, which is critical for resistance to PARP inhibition. Aim 2. Explore strategies to overcome PARPi resistance for breast cancer treatment.